1. Field of the Invention
The present invention relates to the use of skeletal and muscle cardiac cells that continuously secrete recombinant protein products.
2. Discussion of the Background
A large number of inherited and acquired diseases require treatment by intravenous or subcutaneous infusions of proteins. These include inherited protein deficiencies such as hemophilia A and B, and congenital growth hormone deficiency, as well as acquired diseases such as AIDS and diabetes mellitus. Standard therapy for such diseases requires repeated intravenous or subcutaneous administration of protein solutions. The ability to use cell implants which continuously secrete recombinant protein products into the circulation would greatly simplify the therapy of many of these disorders. This disclosure describes transplantable genetically-engineered skeletal muscle stem cells (myoblasts) that produce detectable levels of serum proteins in a recipient host and myocytes that produce local levels of secreted recombinant proteins in the myocardium.
Somatic gene therapy can be defined as the ability to program the expression of foreign genes in non-germ line (i.e., non-sperm and egg) cells of an animal or patient. Recent advances in molecular biology including the cloning of many human genes and the development of viral and chemical gene delivery systems have brought us to the threshold of somatic gene therapy. All methods of gene therapy can be divided into two categories: ex vivo gene therapy involves the removal of cells from a host organism, introduction of a foreign gene into those cells in the laboratory, and reimplantation or transplantation of the genetically modified cells back into a recipient host.
In contrast, in vivo gene therapy involves the introduction of a foreign gene directly into cells of a recipient host without the need for prior removal of those cells from the organism. There are a number of requirements that must be met by any method of gene therapy before it can be considered potentially useful for human therapeutics. First, one must develop an efficient method for introducing the foreign gene into the appropriate host cell. Secondly, it would be preferable to develop systems that program expression of the gene only in the appropriate host cell type, thus preventing expression of the foreign gene in an inappropriate cell. Finally, and most importantly when considering human gene therapy, the technique must have a minimal risk of mutating the host cells and of causing a persistent infection of the host organism, a particularly important worry when using virus vectors to introduce foreign genes into host cells. This application pertains to novel somatic gene therapy for use in heart and skeletal muscle which meets these requirements.
A. In Vivo Gene Therapy in Cardiac Myocytes
The ability to program recombinant gene expression in cardiac myocytes enables the treatment of a number of inherited and acquired cardiac diseases. Therapeutic applications of this approach can be divided into several general categories. First, to correct genetic disorders of myocardial cells. For example, injection of the normal dystrophin cDNA can be used to correct the defects in cardiac contractility seen in patients with Duchenne's muscular dystrophy. Secondly, to stimulate new collateral circulation in areas of chronically ischemic myocardium by injecting plasmids encoding recombinant angiogenesis factors directly into the left ventricular wall. Also, this approach can be used to directly study the molecular mechanisms regulating cardiac myocyte gene expression both during cardiac myogeneses and in a variety of pathophysiologic states such as cardiac hypertrophy.
As many as 1.5 million patients per year in the U.S. suffer a myocardial infarction (MI). Many millions more suffer from syndromes of chronic myocardial ischemia due to large and small vessel coronary atherosclerosis. Many of these patients will benefit from the ability to stimulate collateral vessel formation in areas of ischemic myocardium. The direct DNA injection method provides an alternative approach to the current methods of coronary artery bypass and percutaneous transluminal coronary angioplasty. In particular, many patients have such severe and diffuse atherosclerosis that they are not candidates for CABG or PTCA. Thus far, there has been no approach which has successfully stimulated collateral vessel formation in areas of ischemic myocardium.
A number of genetic disorders affect myocardial performance. For example, many patients with Duchenne's muscular dystrophy also suffer from a cardiomyopathy. In addition, it is clear that there are a number of other genetically-inherited cardiomyopathies of unknown etiology. The gene injection approach described in this disclosure is useful for treating a variety of these inherited disorders of cardiac function. For example, injection of vectors containing the normal dystrophin gene or cDNA can correct the defect in patients with Duchenne's muscular dystrophy. Some aspects of the natural expression of the dystrophin gene in muscle from DMD patients are discussed by Scott et al, Science, 239: 1418 (1988). As additional genes for inherited cardiomyopathy are identified, these gene products might also be injected into hearts in order to correct abnormal cardiac function.
An understanding of the molecular mechanisms that regulate cardiac-specific gene expression during both normal cardiac development and a variety of pathological processes such as cardiac hypertrophy is critical in designing rational therapeutic approaches to such problems. Previous approaches have all utilized in vitro transfection protocols into neonatal cardiocytes or transgenic approaches in mice. Such studies are complicated by the fact that neonatal cardiocytes may not reflect the in vivo situation and by the fact that neonatal cardiocytes have an extremely limited life span in tissue culture and cannot be incorporated into the heart. Moreover, transgenic approaches are lengthy (requiring 6 months to 1 year) technically difficult and expensive. The gene injection approach described in this disclosure obviates these problems because it allows the stable expression of recombinant gene products in both neonatal and adult cardiac myocytes in vivo in as little as 5 days following injection of DNA.
B. Ex Vivo Gene Therapy using Skeletal Myoblasts
A variety of acquired and inherited diseases are currently treated by repeated intravenous or subcutaneous infusions of recombinant or purified proteins. These include diabetes mellitus, treated with subcutaneous or intravenous injections of insulin, hemophilia A, treated with intravenous infusions of factor VIII, and pituitary dwarfism, treated with subcutaneous injections of growth hormone. The development of cellular transplantation systems that can stably produce and deliver such recombinant proteins into the systemic circulation would represent an important advance in our ability to treat such diseases. The ideal recombinant protein delivery system would utilize a cell that can be easily isolated from the recipient, grown and transduced with recombinant genes in vitro, and conveniently reimplanted into the host organism. This cell would produce large amounts of secreted recombinant protein, and following secretion, this protein would gain access to the circulation. Finally, such implanted, genetically engineered cells should survive for long periods of time and continue to secrete the transduced protein product without themselves interfering with the function of the tissue into which they were implanted.
Several different cellular systems have been used to produce recombinant proteins in vivo. These include karatinoxytes (M. Flowers et al, PNAS (USA) 87, 2349 (1990)), skin fibroblasts (T. D. Palmer, A. R. Thompson, A. D. Miller, Blood 73, 438 (1989); R. Scharfmann, J. H. Axelrod, I. M. Verma, PNAS (USA) 88, 4626 (1991)), hepatocytes (D. Armentary, A. Thompson, G. Darlington, S. Woo, PNAS (USA) 87, 6141 (1990); K. P. Ponder et al, PNAS (USA) 88, 1217 (1991)), lymphocytes (K. Culver et al, PNAS (USA) 88, 3155, (1991)), and bone marrow (E. Dzierzak, T. Papayannopoulou, R. Mulligan, PNAS (USA) 87, 439 (1990); M. Kaleko, J. V. Garcia, W. R. A. Osborne, A. D. Miller, Blood 75, 1733 (1990)). Although several of these systems have produced detectable levels of circulating proteins, it has proven difficult to produce stable, physiological levels of circulating recombinant proteins in normal animals.
Burnetti et al, J. Biol. Chem, 265: 5960 (1990) have studied the regulation of myogenin and the events that occur when myoblasts transform into myocytes. However, no expression of a recombinant DNA sequence was disclosed. Paulson et al, J. Cell Biol., 110: 1705 (1990) describes the temperature-sensitive expression of all-torpedo and torpedo-rat hybrid acetylcholine receiptor (AChR in mammalian muscle cells. However, this expression of protein was performed in mouse fibroblasts in culture, and not in vivo. Obtaining expression of physiological serum levels of a protein through a cellular implant is unrelated to and unsuggested by simply obtaining in vitro expression.
Pramanik et al, Eur. J. Biochem., 172: 355 (1988). Showed that translation of P-40 mRNA is repressed in non-proliferating myotubes by using nuclease S1 mapping to quantify the steady-state levels of P-40 mRNA in subcellular fractions of both myoblasts and myotubes. It was shown that the result of the subcellular distribution of this mRNA in proliferating myoblasts following inhibition of DNA synthesis by citazene or arabinoside have shown that translation of P-40 mRNA continued in the absence of DNA synthesis. This observation suggests that an additional signal is necessary to block the translation of P-40 mRNA in myotubes.
Arnold et al (J. Biol. Chem. 257: 9872 (1982)) studied expression of glyceraldehyde-3-phosphate dehydrogenase mRNA in developing chick heart cells in cultures. The gap dehydrogenase mRNA was present in 5 hour old dividing myoblasts. This method is limited to in vitro protein expression and does not address the issue of whether skeletal myoblasts will produce secreted recombinant proteins following differentiation into myotubes.